Assessing site-specific bioavailability is an important consideration in determining and affectively addressing hazards posed by metals-contaminated waste sites. This application addresses the NIEHS Superfund Research Program's needs for fundamental research that improves the understanding of the effects of biogeochemical interactions on contaminant bioavailability. Because metals occur in mixtures rather than as single contaminants at many contaminated sites, accurate assessment of metal exposure and effects to humans and ecosystems must account for mixture effects, which could be additive, less than additive, or more than additive. However, current water quality regulations and management practices usually address individual metals, because adequate tools are not yet available for predicting bioavailability and toxicity of metal mixtures. Our long-term objective isto detect, characterize, and assess the risks posed by contaminant metal mixtures. To accomplish that goal, we need to develop and/or refine advanced techniques for the detection, assessment, and evaluation of metal bioavailability. Those techniques will include environmental molecular diagnostics and stable isotope assays. In the proposed research, the methods we develop under controlled conditions will be field-truthed in a metals-contaminated stream at the North Fork Clear Creek Superfund site in central Colorado. The approaches and tools developed for that site will provide a physical and intellectual decision infrastructure applicable to other meta-contaminated sites. In this project a combination of organism- and community-level response studies (Project 1); genomic bioassays of organism response to multiple-metals exposure (Project 2); and measurements of bioavailable water-column metals and tissue-metal residues (Project 3) will be developed and applied. We will gain an understanding of the flux between biological/chemical/geological interfaces as it relates to bioavailability and remediation effectiveness. Furthermore, we will elucidate the geochemical factors affecting biological uptake (e.g., transport into the food chain). We will also conduct a natural experiment in the metal-contaminated stream in which we will examine responses before, during, and after installation of a water-treatment system that will decrease concentrations of the metals. Using the methods developed and tested during the first two years of the project, we will be able to model and observe the effectiveness of the remediation efforts during the later years of the project. Finally we have delineated a plan for research translation, including engaging end-users (e.g., regulatory agencies, metals-industry representatives, and toxicity modelers) throughout the duration of the grant. Although the focus of the proposed research is a single Superfund site, the methodology and results will be applicable to metal mixtures in many receiving waters, regardless of the contamination source.